BINDING OF COMPLEMENT COMPONENT Clq TO ... - Science Direct

Molecular Immunology, Vol. 34. No. 1, pp. 33-38, 1997 cQ 1997 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161.5890197 $17.00 + 0.00

Pergamon PII: SO1615890(97)00005-9

BINDING OF COMPLEMENT COMPONENT Clq TO MYELIN OLIGODENDROCYTE GLYCOPROTEIN: A NOVEL MECHANISM FOR REGULATING CNS INFLAMMATION TERRANCE Neuroimmunology

G. JOHNS

Laboratory,

and CLAUDE

La Trobe University,

(Received 15 September

C. A. BERNARD*

Bundoora,

Victoria

3083. Australia

1996; accepted 12 December 1996)

Abstract-Myelin oligodendrocyte glycoprotein (MOG) is a myelin-specific protein restricted to the central nervous system (CNS). While MOG is considered a putative autoantigen in MS, its function(s) in myelin is unknown. As CNS myelin is able to activate the classical complement pathway, it must contain a Clq-binding/activating protein but the identity of this protein has not been reported. The data in this paper clearly demonstrate that MOG specifically binds Clq in a dose-dependent and saturating manner. This calcium-dependent interaction is mediated by the extracellular immunoglobulin-like domain of MOG. This MOG domain contains an amino acid motif similar to the core Clq-binding sequence previously identified in IgG antibodies. Purified MOG also inhibited the antibody-dependent lysis of RBC by complement. Taken together, these results demonstrate that MOG binds Clq near the IgG binding site and may be the protein responsible for complement activation in myelin. This direct interaction between a myelin-specific protein and Clq has significant implications for CNS inflammation and could be particularly important in demyelinating diseases such as multiple sclerosis. 0 1997 Published by Elsevier Science Ltd. All rights reserved. Key word.7: myelin, complement,

Clq, neuroimmunology,

INTRODUCTION

contain a protein capable of binding and activating the Clq component of complement. The identity of this protein, which is possibly myelin specific, has not been reported. Examination of the MOG sequence from human, bovine, rat and mouse (Hilton et al., 1995) revealed a conserved motif, E X R X R, at amino acid position 64 in all species. This motif is similar to the core Clq-binding site on IgG molecules (Duncan and Winter, 1988). IgG also contains an upstream N-linked glycosylation site which contributes to Clq binding (Koide et al., 1977). Interestingly, MOG also has a similar upstream glycosylation site at amino acid position 31 (Hilton et al., 1995). Finally, as MOG is expressed predominantly on the outermost lamellae of the myelin sheath, it is fully accessible to Clq. Thus, we tested the hypothesis that MOG is a myelin-specific Clq-binding protein. We report here that the extracellular domain of MOG does display dose-dependent and saturable binding to Clq, in an interaction that requires the presence of calcium. As MOG also inhibited the antibody-dependent complement lysis of target cells, it is possible that MOG is the protein in myelin responsible for the activation of the classical pathway of complement.

Myelin oligodendrocyte glycoprotein (MOG) is a member of the immunoglobulin superfamily found exclusively in central nervous system (CNS) myelin (Gardinier et al., 1992). While this protein (Mr 28 000) has been identified as a potential autoantigen in multiple sclerosis (Kerlero de Rosbo et al., 1993; Johns et al., 1995), its function remains unknown. Given that MOG is expressed late in myelin development (Scolding et al., 1989a), it has been suggested that it has a role in the completion and/or compaction of the myelin sheath, although there is no direct evidence supporting this view. The localization of the MOG gene within the MHC (Pham-Dinh et al., 1993), its homology to the B-G antigens of the chicken MHC (Gardinier et al., 1992). its structural similarity to the B7 gene family (Linsley et al., 1994) and its ability to elicit strong immune responses (Linington and Lassman, 1987) have led some to speculate that MOG may have an immune function within the CNS (Linsley et al., 1994; Steinman, 1993). Purified CNS myelin, but not peripheral nervous system myelin, can activate the classical pathway of complement (Vanguri and Shin, 1986). Furthermore, mature rat oligodendrocytes grown in vitro are lysed by complement in the absence of antibody (Scolding et al., 19896; Wren and Noble, 1989). Therefore, CNS myelin must

*Author

to whom correspondence

brain.

MATERIALS AND METHODS Reagents

The Fabz fragment of the mouse monoclonal antiMOG antibody. 8-18C5, was a gift from Dr Sarah

should be addressed. 33

34

T. G. JOHNS

and C. C. A. BERNARD

Piddlesden (University of Wales, Cardiff, U.K.). The fragment was biotinylated using a biotinylation kit from Amersham (Sydney, Australia) according to the manufacturer’s instructions. Purified Clq was purchased from Calbiochem (Melbourne, Australia). Goat anti-Clq serum and Clq-deficient serum were both purchased from Sigma (St Louis, MO, U.S.A.). Preparation

RESULTS AND DISCUSSION

qf MOG and MBP

Native rat and human MOG were prepared as previously described in detail (Abo et al., 1993). Purified MOG was stored at - 70’C in 25 mM Tris, pH 7.4. The recombinant extracellular domain of human MOG (rMOG) was expressed in E. coli as a GST fusion protein (Jayaram et al., 1996; Ichikawa et al., 1996). Recombinant MBP was prepared as described by Oettinger et al.. 1993. Binding

qfMOG

to Clq-coated ELISA

plates

Plates were coated with 100 ~1of purified Clq (10 pg/ml unless otherwise stated) in PBS for 90 min at 37°C. After washing with 0.1% Tween in PBS (PBS-T) plates were blocked with 2% BSA. MOG diluted in PBS (lO,ug/ml unless otherwise stated) was then added to the wells for 60min at 37°C. MOG bound to the Clq was detected using the biotinylated Fab, fragment of 8- 18C5 at a concentration of 0.1 pg/ml in 2% BSA. After incubation with streptavidin-horseradish peroxidase (Amersham, Sydney, Australia), specific binding was determined using 2.2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as a substrate and the absorbance measured at 405 nm. Binding of the Fab, fragment directly to Clq was negligible. In some experiments, the binding of MOG was done in the presence of 10 mM EDTA or EGTA. This level of EDTA or EGTA had no effect on the amount of Clq bound to the plate as measured using anti-Clq goat serum. Binding

RBC (12 x 10’ cells/ml) previously incubated with a 1: 1000 dilution of rabbit anti-sheep RBC (Serotee, Oxford, U.K.). After incubation at 37°C for 15 min, cell lysis was measured by determining the supernatant absorbance at 414 nm as recommended by the supplier (Sigma).

qf Clq to MOG-coated

Purified human MOG bound Clq-coated ELISA plates, with the level of binding showing excellent correlation to the amount of Clq bound to the plate (Fig. 1). Indeed, the ratio between MOG binding and the amount of Clq bound to the plate remained constant at all concentrations tested, suggesting a specific interaction between the two proteins. Using a fixed concentration of Clq, human MOG displayed a dose-dependent and saturating binding curve (Fig. 2(A)), which is highly indicative of specific binding. A similar result was obtained with purified rat MOG (Fig. 2(B)), showing that this interaction is species independent. This observation was not unexpected, given the high homology between MOG from different species (Hilton et al., 1995) and the conservation of the proposed Clq-binding site. The recombinant extracellular domain of MOG was purified as a GST fusion protein from E. coli. This protein also bound Clq in a dose-dependent manner (Fig. 2(C)), confirming that the MOG Clq-binding site is located within the extracellular domain and not in the hydrophobic transmembrane regions or the cytoplasmic domain. The seemingly lower binding of the rMOG may reflect conformational differences, a lower affinity for the 8-18C5 antibody used for detection or the lack of glycosylation which is important for stabilizing antibody-Clq interactions (Koide et al., 1977). The interaction between

1.2-

ELISA

Plates were coated with 100~1 MOG or myelin basic protein (MBP) (10 pg/ml) in carbonate buffer, pH 9.6 for 90min at 37°C. After washing with PBS-T, plates were blocked with 2% BSA. Clq (2.5 Llg/ml) in PBS with 1 mM Ca’+ was then added and incubated for 60min. Bound Clq was detected using anti-Clq goat serum (1:5000) followed by a 1:20 000 dilution of anti-goat conjugated to horseradish peroxidase (Silenus, Melbourne, Australia). After the addition of ABTS substrate the absorbance was measured at 405 nm. Assayfbr

complement

-C

anti-MOO

-o-

antic1q

plates

cell lysis

Purified Clq (1 pg) was pre-incubated for 15min at 37°C with 300 ~1of Verona1 buffer, pH 7.2 (145 mM NaCl, 1 mM Mg2+ and 0.2 mM Ca’+), or with buffer containing 2.5pg of MOG or MBP. Clq-deficient serum (lop), reconstituted with Mg’+ and Ca’+ according to the supplier’s instructions (Sigma), was added to the Clq. The now complete serum was then added to 200~1 of sheep

0

5

10

Cl q concentration

15

20

(pg./ml)

Fig. I. Binding of purified human MOG to Clq. Clq in PBS was coated on an ELISA plate at the indicated concentrations. The binding of MOG (lO,ug/ml) was determined using a biotinylated 8-18C5 Fabz antibody fragment as described in Materials and Methods. Absorption of Clq to the ELISA plate was measured using anti-Clq goat serum. The data represent the mean + SE of triplicate determinations.

Myelin oligodendrocyte

35

glycoprotein binds Clq

0.8 P 80.8 3 f 0.4 fn 9

0.2 0.0

0

0

10 15 20 25 5 Native Human MOG (pg/ml)

5 7.5 2.5 Native Rat MOG (pg/ml)

10

0

20 10 Recombinant

30 40 MOG @/ml)

50

Fig. 2. Binding of native and recombinant MOG to Clq. ELISA plates were coated with lOpg/ml Clq (m) or 2% BSA (@) and the binding of increasing concentrations of human MOG (A), rat MOG (B) or rMOG (C) was determined as described in Fig. 1. The data represent the mean + SE of triplicate determinations.

MOG and Clq was not simply a non-specific interaction between a surface-bound monolayer of Clq and MOG, because the addition of increasing amounts of Clq in solution led to a progressive decrease in the amount of human MOG which bound to Clq-coated ELISA plates (Fig. 3). This result implies that MOG can form solution complexes with Clq. The addition of 1OmM EDTA or EGTA during the MOG binding step reduced the binding of human MOG to Clq by over 90% (Fig. 4(A)). This result demonstrates that calcium is essential for binding and further confirms the specificity of the Clq-MOG interaction, as it is unlikely that the addition of 1OmM EDTA or EGTA

3

~0.8 8 s 0.6

e

8 9 0.4

0.2 1

o.o-, 0

5

10

Cl q concentration

15

20

(pg/ml)

Fig. 3. The effect of Clq in solution on the binding of MOG to immobilized Clq. Clq (lOpg/ml) in PBS was coated on an ELISA plate. The binding of human MOG (10 pg/ml) to the immobilized Clq, in the presence of soluble Clq at the indicated concentrations. was then measured as in Fig. 1. The data represent the mean rt SE of triplicate determinations.

would block a non-specific interaction. It should also be noted that EDTA or EGTA had no effect on the level of Clq bound to the ELISA plate (data not shown). MOG previously dialysed against TBSjl mM Ca2+ was diluted into TBS or TBS containing 5 mM of either Ca”, Mg2+, Mn’+ or Zn’+ (Fig. 4(B)). The addition of Ca’+ at higher concentrations (4.5 mM final concentration) enhanced the binding of MOG to Clq two-fold when compared to buffer alone (final concentration 0.13 mM), further demonstrating the requirement for Cal-. The addition of Mg’+ had no effect on binding when compared to buffer alone, whereas both Mn’+ and Zn2+ inhibited the binding of MOG to Clq (Fig. 4(B)). It is interesting that the binding of Clq to the two serine proteases, Clr and Cls, as well as to the gp41 protein from HIV are also calciumdependent interactions, although they involve different binding motifs (Thielens et al., 1990; Stoiber et al., 1995). It is not possible from our experiments to determine if the calcium is required by Clq, MOG or both. The binding of IgG to the globular domain of Clq does not require the presence of calcium (Duncan and Winter, 1988). As we are proposing that MOG interacts with Clq in a similar manner to IgG. it is tempting to speculate that the calcium is required by MOG and not Clq. It is possible that the binding of calcium to MOG may induce the confirmation changes necessary for its binding to Clq. If MOG does bind calcium, then the results in Fig. 4(B) suggest that multiple divalent ions also are capable of binding MOG. Preliminary work in our laboratory suggests that MOG is a calcium-binding protein as the binding of some MOG specific antibodies is also calcium dependent (T. G. Johns, unpublished observations). To further confirm the specificity of the MOG-Clq interaction, the binding of Clq to MOG- or MBP-coated ELISA plates was compared. MBP was chosen as it is another myelin specific protein that shares some properties with MOG, in that it is highly basic and contains a number of hydrophobic regions (Oettinger et al.. 1993). While Clq bound to the MOG-coated plates. the binding

36

T. G. JOHNS and C. C. A. BERNARD

0.8 T 1.6 c

p 0.7 ;

3 3 1.2

0.6

SO.5 8 s 0.4

8 :s 0.8

3 0.3 2 0.2

9 0.4

PBS

EDTA

EGTA

Fig. 4. (A) Binding of human MOG to Clq in the presence of chelators. Clq (lOpg/ml) in PBS was coated on an ELISA plate. The binding of human MOG (10 fig/ml) was then compared in the presence or absence of EDTA or EGTA (10mM) as described in Fig. 1. The data represent meanf SE of triplicate determinations. (B) Binding of human MOG to Clq in the presence of divalent ions. Human MOG (50 pg/ml) in TBSjl mM Ca’+ was diluted to 6.7 pg/ml in TBS alone or TBS containing either 5mM Ca’+. Mg’+, Mn’+ or Zn’+. Binding of MOG to Clq was then compared as described in Fig. 1. The data represent mean k SEM of triplicate determinations.

to MBP was similar to that observed for BSA alone (Fig. 5), further supporting the notion that MOG is a myelin protein that specifically interacts with Clq. In order to determine if the interaction between MOG and Clq is biologically significant, we tested the ability of MOG to inhibit the haemolytic activity of complement towards RBC. Punified Clq (1 pg) was pre-incubated with purified MOG, MBP or buffer alone and then used to reconstitute Clq-deficient serum. Sheep RBC, pretreated with rabbit anti-sheep RBC anti-serum, was then added and cell lysis determined using a standard complement assay. While MBP had no effect on RBC lysis, purified MOG was able to inhibit lysis by 76% (Fig. 6). This indicates that, as predicted, MOG binds Clq at, or in

close proximity to, the IgG recognition site and suggests that the binding of Clq to MOG could activate the classical complement pathway. Our data clearly demonstrate a specific interaction between Clq and MOG. a quantitative minor component of myelin expressed on the outermost layer of the myelin sheath (Gardinier et al., 1992) The Clq-binding domain of MOG is restricted to the extracellular IgG-like region. As this region contains the core amino acid motif (E X R X R), shown to be important for the binding of IgG 1.2

1.0 g = 0.8 t 5 8 0.6 2 1 0.4

a

0.2 0.0

WOG MBP BSA Fig. 5. Comparison of Clq binding to MOG and MBP. ELISA plates were coated with human MOG (10 pg/ml) or human MBP (lOpg/ml) in carbonate buffer pH 9.6. The binding of Clq (2.5pg/ml) was measured using anti-Clq goat serum as described in Materials and Methods. The data represent mean k SE of triplicate determinations.

Control MBP MOG Fig. 6. MOG inhibits antibody-mediated complement cell lysis. Clq-deficient serum (10 ~1) was reconstituted with purified Clq (1 pg) pre-incubated with buffer alone, native human MOG (2.5 pg) or MBP (2.5 pg) as described in Materials and Methods. The serum was then added to sheep RBC pretreated with rabbit anti-sheep RBC serum. Cell lysis was determined after 15 min by measuring haemoglobin at an absorbance of 414nM. The data represent mean f SE of triplicate determinations.

Myelin oligodendrocyte to the globular

domain of Clq (Duncan and Winter, 1988), it is likely that MOG binds to the IgG binding domain of Clq. This contention is further supported by the observation that MOG is able to block the antibodydependent lysis of RBC and preliminary data showing that aggregated human IgG blocks the binding of MOG to Clq (Johns, unpublished observations). Thus, MOG is not only a myelin-specific Clq-binding protein, but is probably responsible for the activation of complement by CNS myelin, although we cannot exclude the possibility that other myelin components are able to bind Clq. Significantly, peripheral myelin which lacks MOG, but contains many other proteins in common with CNS myelin, is unable to activate the classical complement pathway (Vanguri and Shin. 1986). The binding of Clq to rat oligodendrocytes leads to complement activation and cell lysis (Scolding et al., 1989b; Wren and Noble, 1989). The sensitivity of rat oligodendrocytes to complement is not only the result of Clq binding but the lack of CD59 (Wing et ul., 1992; Piddlesden and Morgan, 1993). a molecule that prevents formation of terminal attack complexes. The incorporation of CD59 into the membrane of rat oligodendrocytes prevents their lysis by complement (Wing et ul., 1992). While both human myelin and oligodendrocytes bind Clq and probably activate complement to at least the C3 stage (Vanguri and Shin, 1986; Zajicek et al., 1995) human oligodendrocytes are not lysed by complement as they contain CD59 (Zajicek er al., 1995). Since Clq binding, and the partial activation of complement by myelin. is conserved between species, it may well have a beneficial function. For example, it may be important in the opsonization and subsequent removal of myelin debris. the destruction of damaged oligodendrocytes or the amplification of CNS immune responses. Furthermore, the rapid increase in Clq level that occurs as the result of brain inflammation (Dietzschold et al., 1995) could stimulate a variety of oligodendrocyte responses following binding of Clq to the cell. Indeed, recent results demonstrating that sublytic complement levels induce cell cycle in oligodendrocytes support such a proposal (Rus et al., 1996). Thus. binding of Clq, or other complement components, to oligodendrocytes may prime these cells for the remyelination of damaged areas, once the inflammation has subsided. Alternatively, the binding of Clq to myelin and oligodendrocytes, or the subsequent activation of complement, may be dysfunctional in some inflammatory diseases of the CNS. Therefore, inhibition of the ClqMOG interaction may represent a novel therapeutic target for CNS inflammatory diseases including multiple sclerosis. where complement activation has a significant role (Shin and Koski. 1992). Indeed, the high level of antibody seen in the CSF of most multiple sclerosis patients might be a mechanism which the body uses to prevent excessive complement activation in the CNS. This hypothesis is supported by the observation that exogenous IgG is beneficial to some multiple sclerosis patients (Miller et ul., 1995) and the recent report showing that purified IgG is able to prevent complement-mediated

31

glycoprotein binds Clq

hyperacute rejection in xenotransplantation (Magee et al., 1995) Thus, it is tempting to speculate that inhibition of the Clq-MOG interaction could be a mechanism for reducing excessive inflammation in a variety of conditions including chronic viral infections and neurodegenerative diseases. In conclusion, this study is the first report of an interaction between a myelin-specific protein and an important component of the immune system. We believe this observation has important implications with regard to the mechanisms regulating CNS inflammatory responses. Acknowledgernenrs-We thank MS Sharon Harris for her expert secretarial assistance, Dr Sarah Piddlesden for the Fab, fragment of 818C5, and Dr S.B. Easterbrook-Smith (University of Sydney, NSW, Australia) for his advice on complement ELISAs. The work was supported by the National Health and Medical Research Council of Australia (NH and MRC). Dr Terrance G. Johns is the recipient of an NH and MRC Postdoctoral Fellowship.

REFERENCES Abo S.. Bernard C. C. A., Webb M., Johns T. G.. Alafaci A., Ward L. D., Simpson R. J. and Kerlero de Rosbo N. (1993) Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies. Biochem. Molec. Biol. Int. 30, 945-958. Dietzschold B., Schwaeble W., Schafer M. K. H., Hooper D. C.. Zehng Y. M., Petry F.. Sheng H., Fink T., Loos M., Koprowski H. and Weihe E. (1995) Expression of Clq, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental allergic encephalomyehtis. J. Neural. Sri. 130, 11-I 6. Duncan A. R. and Winter G. (1988) The binding site for Clq on IgG. Nature 332, 738-740. Gardinier M. V., Amiguet P., Linington G. and Matthieu J.-M. (1992) Myelin/oligodendrocyte glycoprotein is a unique member of the immunoglobulin superfamily. .I. Neurosci. Res. 33, 177-I 87. Hilton A. A., Slavin A. J.. Hilton D. J. and Bernard C. C. A. (1995) Characterisation of cDNA and genomic clones encoding human myelin oligodendrocyte glycoprotein. J. Neurochem. 65, 309-3 18 Ichikawa M., Johns T. G.. Adelman M. and Bernard C. C. A. (1996) Antibody response in Lewis rats injected with myelin ohgodendrocyte glycoprotein derived peptides. ht. Inzmun.. 8, 166771674. Jayaram B. M.. Menon K. K., Barton T.. Slavin A., Reid H. and Bernard C. C. A. (1996) Purification of human myelin

oligodendrocytes glycoprotein (MOG) produced in bacteria and St9 insect cells. J. Neurochem. 66(Suppl. 2) S41. Johns T. G.. Kerlero de Rosbo N., Menon K. K., Abo S., Gonzales M. F. and Bernard C. C. A. (1995) Myelin oligodendrocyte glycoprotein induces a demyehnating encephalomyelitis resembling multiple sclerosis. J. l~rrn~~~z.154, 5536 5541. Kerlero de Rosbo N., Milo R., Lees M. B., Burger D., Bernard C. C. A. and Ben-Nun A. (1993) Reactivity to myelin antigens in multiple sclerosis. J. Clin. Iwest. 92, 2602-2607. Koide N.. Nose M. and Muramatsu T. (1977) Recognition of IgG by Fc receptor and complement: effects of glycosidase digestion. Biochern. Biolphys. Res. Commtm. 75, 838-844.

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T. G. JOHNS

and C. C. A. BERNARD

Linington C. and Lassman H. (1987) Antibody responses in chronic relapsing experimental allergic encephalomyelitis: correlation of serum demyelinating activity with antibody titre to the myelin/oligodendrocyte glycoprotein (MOG). J. Neuroimmun. 17,61-69. Linsley P. S., Peach R., Gladstone P. and Bajorath J. (1994) Extending the B7 (CD80) gene family. Protein Sci. 3, 13411343. Magee J. C., Collins B. H., Harland R. C., Lindman B. J., Bollinger R. R., Frank M. M. and Pratt J. L. (1995) Immunoglobulin prevents complement-mediated hyperacute rejection in swine-to-primate xenotransplantation. J. Clin. Inwst. 96, 240424 12. Miller D. J., Asakura K. and Rodriguez M. (1995) Experimental strategies to promote central nervous system remyelination in multiple sclerosis: insights gained from the Theiler’s Virus model system. J. Neurosci. Res. 41, 291-296. Oettinger H. F., Al-Sabbagh A., Jingwu Z., LaSalle J. M., Weiner H. L. and Hafler D. A. (1993) Biological activity of recombinant human myelin basic protein. J. Neuroimmun. 44, 157-I 62. Pham-Dinh D.. Mattei M.-G., Nussbaum J.-L., Roussel G.. Pontarotti P., Roeckel N., Mather I. H.. Artzt K., Lindahl K. F. and Dautigny A. ( 1993) Myelin/oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex. Proc. natn. Acad. Sci. U.S.A. 90, 7990-7994. Piddlesden S. J. and Morgan B. P. (1993) Killing of rat glial cells by complements deficiency of the rat analogue of CD59 is the cause of oligodendrocyte susceptibility to lysis. J. Neuroimmun. 48, 169-l 76. Rus H. G.. Niculescu F. and Shin M. L. (1996) Sublytic complement attack induces cell cycle in oligodendrocytes. J. Immun. 156, 48924900. Scolding N. J., Frith S., Linington C., Morgan B. P., Campbell A. K. and Compston D. A. S. (1989a) Myelin-oligodendrocyte glycoprotein (MOG) is a surface marker of oligodendrocyte maturation. J. Neuroimmun. 22, 1699176.

Scolding N. J., Morgan B. P., Houston A., Campbell A. K., Linington C. and Compston D. A. S. (1989b) Normal rat serum cytotoxicity against syngeneic oligodendrocytes complement activation and attack in the absence of anti-myelin antibodies. J. Neurol. Sci. 89,289-300 Shin M. L. and Koski C. L. (1992) The complement system in demyelination. In Mveli?z Biology? and Chemistry (Edited by Martenson, R. E.), p. 801, CRC Press, Boca Raton, FL. Steinman L. (1993) Connections between the immune system and the nervous system. Proc. natn. Acad. Sci. U.S.A. 90, 7912-7914. Stoiber H., Ebenbichler C. F., Thielens N. M., Arlaud G. J. and Dierich M. R. (1995) HIV-l rsgp41 depends on calcium for binding of human Clq but not for binding of gp 120. Mol. Immun. 32, 371-374. Thielens N. M., Aude C. A., Lacroix M. B., Gagnon J. and Arlaud G. J. (1990) Ca’+ binding properties and Ca” + dependent interactions of the isolated NH>-terminal fragments of human complement proteases Clr and Cls. J. Biol. Chem. 265, 14469914475. Vanguri P. and Shin M. L. (1986) Activation of complement by myehn: identification of Cl-binding proteins of human myelin from central nervous tissue. J. Neurochem. 46, 15351541. Wing M. G., Zajicek J. P., Seilly D. T., Compston D. A. S. and Lachmann P. J. (1992) Oligodendrocytes lack glycolipid anchored proteins which protect them against complement lysis. Restoration of resistance to lysis by incorporation of CD59. Immunologic 76, 140-155. Wren D. R. and Noble M. (1989) Oligodendrocytes and oligodendrocyteitype-2 astrocyte progenitor cells of adult rats are specifically susceptible to the lytic effects of complement in the absence of antibody. Proc. Ilatn. Awd. Sci. U.S.A. 86, 9025s9029. Zajicek J., Wing M., Skepper J. and Compston A. (1995) Human oligodendrocytes are not sensitive to complement. Lab. bwest. 73, 128-138.